A novel anti-tumor agent, Ln(III) 2-thioacetate benzothiazole induces anti-angiogenic effect and cell death in cancer cell lines

Article abstract of DOI:10.1016/j.ejmech.2012.02.025

New complexes with a potent DNA-binding anti-tumor agent, europium(III)- and terbium(III)-2-thioacetate benzothiazole were synthesized and characterized. These complexes showed strong binding affinity to calf thymus DNA using fluorometric and electronic absorption spectroscopy. The synthesized complexes resulted in inhibition of proliferation of EAC cells and ascites formation. Their anti-tumor effect was found to be through anti-angiogenic activity as was evident by the reduction of microvessel density and down-regulation of VEGF receptor type-2 (Flk-1). It was found that EAC cells had distinct DNA fragmentation patterns analyzed by capillary electrophoresis in the treated animals. Moreover, the synthesized complexes exhibited significant cytotoxic activity against HepG2 and MCF7 cell lines. Furthermore, complexes showed a potent anti-bacterial activity against two pathogenic bacteria Escherichia coli and Salmonella.

Full text of DOI:10.1016/j.ejmech.2012.02.025

European Journal of Medicinal Chemistry 51 (2012) 99e109
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
A novel anti-tumor agent, Ln(III) 2-thioacetate benzothiazole induces
anti-angiogenic effect and cell death in cancer cell lines
,
Belal H.M. Hussein a, Hassan A. Azab a, Mona F. El-Azab b, Abdullah I. El-Falouji c
*
a Chemistry Department, Faculty of Science, Suez Canal University, Ismailia 41522, Egypt
b Department of Pharmacology and Toxicology, Faculty of Pharmacy, Suez Canal University, Ismailia 41522, Egypt
c Biotechnology Research Center, Suez Canal University, Ismailia 41522, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Newcomplexes with a potent DNA-binding anti-tumor agent, europium(III)- and terbium(III)-2-thioacetate
benzothiazole were synthesized and characterized. These complexes showed strong binding affinity to calf
thymus DNA using fluorometric and electronic absorption spectroscopy. The synthesized complexes
resulted in inhibition of proliferation of EAC cells and ascites formation. Their anti-tumor effect was found to
be through anti-angiogenic activity as was evident by the reduction of microvessel density and down-
regulation of VEGF receptor type-2 (Flk-1). It was found that EAC cells had distinct DNA fragmentation
patterns analyzed by capillary electrophoresis in the treated animals. Moreover, the synthesized complexes
exhibited significant cytotoxic activity against HepG2 and MCF7 cell lines. Furthermore, complexes showed
a potent anti-bacterial activity against two pathogenic bacteria Escherichia coli and Salmonella.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Received 27 November 2011
Received in revised form
11 February 2012
Accepted 13 February 2012
Available online 21 February 2012
Keywords:
Lanthanum complex
2-Thioacetic acid benzothiazole
Cytotoxicity
Antitumor activity
Antibacterial activity
DNA fragmentation
1. Introduction
electron transfer reactions, DNA footprinting, and sequence-
specific cleaving agents [19e22]. Of these studies, the interaction
Although struggling efforts against cancer have grown
tremendously in the last few years, cancer is still the second leading
cause of death in economically developed countries following heart
disease. 7.1 millions of people died in 2007 of cancer worldwide
and it is expected that approximately 12 million cancer patients
will die by 2015 [1,2]. Therefore, the discovery and development of
novel therapeutic agents for the treatment of cancer have a vital
importance. It has been reported that lanthanides, as a group of
therapeutic rare earth complexes, manifest anti-tumor activity
[3e12]. Hence, lanthanides represent an effective approach for
searching novel anti-tumor agents. DNA is one of the most
important pharmacological targets of many drugs currently in
clinical use. Drugs that bind with genomic DNA or RNA have proven
to be effective anti-tumor, antivirus and anti-bacterial therapeutic
agents [13e15]. Therefore, the interactions of metal complexes
with DNA have been the subject of interest for the development of
anticancer drugs [16e18] and effective chemotherapeutic agents
for numerous diseases, probes for nucleic acids, DNA-dependent
of fluorescent metal complexes, containing multidentate aromatic
ligands, with DNA has gained much attention [23,24].
Benzothiazole are one of sulfurand nitrogen-containing aromatic
heterocyclic compounds, which are formed from fusion of the aryl
and thiazolyl rings. They have diverse interesting medical and
industrial applications [25]. Many studies on the synthesis and anti-
tumor activity of benzothiazole derivatives displayed a potent and
a selective anti-tumor activity against different types of tumors such
as breast, ovarian, lung, renal cell lines, and colon cancer. Moreover,
those studies also demonstrated that benzothiazole derivatives have
antimicrobial, antitubercular, anti-inflammatory, antirheumatic,
and antiglutamate activities [26e34].
In this work, novel Eu (III) and Tb(III) complexes containing
ligand of 2-thioacetate benzothiazole were synthesized and char-
acterized. The interaction of europium (III)- or terbium (III)-2-
thioacetate benzothiazole with double stranded calf thymus DNA
(ct-DNA) was investigated by fluorescence and UV spectroscopic
techniques. Furthermore, the anti-tumor and anti-angiogenic
effects of these complexes against Ehrlich ascites carcinoma (EAC)
cells, cytotoxicity against HepG2 and MCF7 human tumor cell lines
and anti-bacterial activity against two pathogenic bacteria were
also tested.
* Corresponding author. Tel.: þ2 0115990299; fax: þ2 064 3200452.
E-mail address: falouji@hotmail.com (A.I. El-Falouji).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.02.025

100
B.H.M. Hussein et al. / European Journal of Medicinal Chemistry 51 (2012) 99e109
2. Experimental
N
S
ClCH2COOH
N
S
SCH2COOH
SH
2.1. Materials and reagents
KOH
TbCl3.6H2O, EuCl3.6H2O, RPMI-1640 media and calf thymus DNA
were purchased from Sigma (St. Louis, MO, USA) and used without
further purification. GF-1 DNA Extraction Kit was purchased from
Vivantis. Primary antibody Flk-1 was purchased from Santa Cruz
Biotechnology (CA, USA) and anti-CD31 Class II was purchased from
Dako Cytomation (Carpinteria, USA). HANK’s balanced salt solution
and fetal bovine serum (FBS) were purchased from Invitrogen Inc.
(Carlsbad, CA, USA). Double deionized water was used for all
experiments. The stock solution of calf thymus deoxyribonucleic
acid (ct-DNA) was prepared in TriseHCl buffer solution at pH 7.4
and stored in refrigerator at 4 ꢀC until used. DNA concentrations
were determined by Nano-Drop spectrophotometer at the wave-
length of 260 nm, a solution of ct-DNA in the buffer gave a ratio of
UV absorbance of about 1.89 at 260 and 280 nm, indicating that the
DNA was sufficiently free from protein [35,36].
LnCl3. 6H2O
Et3N
O
O
N
O
O
S
N
SCH2
Ln
CH2S
S
Ln= Eu(III) and Tb(III)
Scheme 1. Synthesis of 2-thioacetic acid benzothiazole ligand and Ln(III)-2-
thioacetate benzothiazole complexes.
Rt 4.20 min. The mobile phase was prepared by mixing acetonitrile
and 25 mM NaH2PO4 in a ratio of 50:50 (v/v). The chemical struc-
ture of 2-thioacetic acid benzothiazole was confirmed by elemental
analysis and spectroscopic methods. 2-Thioacetic acid benzothia-
zole was obtained as a yellowish white solid. Yield 80%; mp:
2.2. Cell lines
Human liver carcinoma (HepG2), human breast carcinoma
(MCF7) and Ehrlich ascites carcinoma (EAC) cell lines were
purchased from the Tumor Biology Department, National Cancer
Institute, Cairo University (Cairo, Egypt). HepG2 and MCF7 were
maintained in RPMI-1640 supplemented with 10% FBS and 1% pen/
145e147 ꢀC; IR (KBr,
1H NMR (CDCl3):
(ppm) ¼ 10.57 (br, 1H, COOH),7.20- 7.9 (4H, m,
ArH), 4.17 (2H, s, SCH2; 13C NMR (CDCl3):
n ;
/cmꢁ1) 3435, 1710, 1573, 1031 and 693 cmꢁ1
d
d
(ppm) ¼ 169.79 (C]O),
151.09 (N]C(S)-S, Cq), 134.87 (N-C,Cq), 126.86 (S-C,Cq), 125.42
(CH Ar), 124.54 (CH Ar), 121.54 (CH Ar), 121.16 (CHAr), 35.8 (S-CH2);
MS (m/z): 225.29 (Mþ.); Anal. Calcd. for C9H7NO2S2 : C, 47.98; H,
3.13; N, 6.22; S, 28.1. Found: C, 49.0; H, 3.40; N, 6.23; S, 28.4.
strep (100 units/ml penicillin and 100
mg/ml streptomycin). The
cells were grown at 37 ꢀC in a humidified incubator containing 5%
CO2 in air. Cells were passaged biweekly and used for experiments
in the exponential growth phase. EAC is a murine spontaneous
breast cancer from which an ascites variant was obtained. The
tumor cell line was maintained in our laboratory by serial intra-
peritoneal (i.p.) passage in female Swiss albino mice at 7e10 day
interval. Tumor cells were tested for viability and contamination
using trypan blue dye exclusion technique [37].
2.5. Synthesis and characterization of Ln(III) 2- thioacetate
benzothiazole
2-Thioacetic acid benzothiazole (1.0 mmol, 0.224 g) was dis-
solved in ethanol (20 mL) and triethylamine (1.0 mmol, 0.102 g) was
then added. The solution was stirred for 15 min at room tempera-
ture. LnCl3.6H2O (0.5 mmol) dissolved in 20 ml ethanol and then
added slowly with vigorous stirring to the 2-thioacetic acid ben-
zothiazole solution. Precipitate appeared immediately after mixing
the two solutions. Stirring overnight has been performed to
complete precipitation. This product was collected by filtration,
purified by washing several times with hot ethanol and dried at
50 ꢀC. The suggested structure of Eu(III)- and Tb(III)-complexes
shown in Scheme 1 was confirmed by IR and mass spectroscopic
methods and elemental analysis. Yield: 55%; Color: yellowish
2.3. Apparatus
Fluorescence spectra were recorded on a Jasco FP-6300 spec-
trofluorometer with a 150W xenon lamp for excitation. Absorbance
spectra were recorded on Jasco FP-6300 attached UV unit using
a 1.0 cm path length cell. Nanodrop spectrophotometer model ND
1000 was used for measuring DNA concentrations. Multi-capillary
electrophoresis QIAxcel (QIAGEN Germany) system was used for
DNA fragmentation analysis. The infrared spectra were obtained in
the 4000e400 cmꢁ1 region by using Bruker Alpha with KBr discs.
Mass spectrometry was carried out by Shimazdu Qp-2010 plus
mass analyzer, 1H and 13C-NMR spectra were recorded on a Joel-
400 NMR, elemental analysis was carried out by elementar vario.
brown; mp: over 300 ꢀC; IR (KBr, /cmꢁ1): 3345, 1732, 1705, 1623,
n
1558, 1028, 689 and 515 cmꢁ1; MS (m/z): 780.1 (Mþ.); Anal. Calcd.
for[Eu(TAB)2Cl.3H2O].5H2O: C, 27.66; H, 3.69; N, 3.58; S, 8.22;
Eu(III), 19.48. Found: C, 28.3; H, 3.29; N, 4.09; S, 7.61; Eu(III), 19.2.
Complex [Tb (TAB)2Cl.3H2O].5H2O was synthesized as described
above, Yield: 40%; Color: yellowish brown. mp: over 300 ꢀC; IR (KBr,
2.4. Synthesis and characterization of 2-thioacetic acid
benzothiazole
n
/cmꢁ1): 3332, 1709, 1627, 1563, 1453, 1033, 694 and 530 cmꢁ1; MS
(m/z):787(Mþ.); Anal. Calcd. for[Tb(TAB)2Cl.3H2O].5H2O: C, 27.47;
H, 3.85; N, 3.56; S, 8.15; Tb(III), 20.19. Found: C, 26.9; H, 3.99; N,
4.03 S, 8.0; Tb(III), 19.8.
2-Thioacetic acid benzothiazole was prepared according to the
method in literature [38,39]. Commercially available 2-mercapto
benzothiazole (0.1 mol) was added to a solution of chloroacetic
acid (0.1 mol) in aqueous methanol in the presence of KOH as
a basic media. The reaction mixture was refluxed for 4 h, then
cooled, filtered and acidified with diluted hydrochloric acid. The
formed precipitate after filtration was crystallized from aqueous
ethanol to give (80%) of the 2-thioacetic acid benzothiazole. The
steps of the synthesis of 2-thioacetic benzothiazole were displayed
in Scheme 1. The purity of 2-thioacetic acid benzothiazole was
confirmed by RPHPLC. Only one sharp peak appears at 300 nm with
2.6. In vitro anti-tumor activity
The effect of europium (III) and terbium (III)-2-thioacetate
benzothiazole complexes on the growth of HepG2 and MCF7 cell
lines were evaluated according to the procedure adopted by the
National Cancer Institute for in vitro anti-tumor drug screening that
using Skehan method [40]. Cisplatin was used as a standard to
assess the cytotoxicity of complexes. The capabilities of complexes

B.H.M. Hussein et al. / European Journal of Medicinal Chemistry 51 (2012) 99e109
101
to arrest the proliferation of tumor cells were evaluated after 48 h
of incubation. The results were analyzed by means of cell viability
curves and expressed as IC50 values. Cells were plated in 96-well
plate (104 cells/well) for 24 h by using RPMI-1640 media before
treatment with the compounds to allow attachment of cell to the
wall of plate, different concentrations of the compound under
electrophoresis according to the protocol AM320 (applied separa-
tion time: 323 s, method separation time: 320 s, method injection
time: 10 s, method separation voltage: 6.0 kV and method injection
voltage: 5.0 kV) of the QIAxcel DNA Screening Kit.
2.7.6. Immunohistochemistry
test (1, 5, 10, 25 and 50
m
M) were added to the cell monolayer
IHC was performed on 5-um paraffin sections using the avidin-
biotin-peroxidase method. Sections were deparaffinized in xylene
and rehydrated in a graded series of ethanol and rinsed with
distilled water. Antigen retrieval was performed by citrate-EDTA
antigen retrieval protocol [46]. Endogenous peroxidase activity
was blocked by incubation in 3% hydrogen peroxide for 5 min.
Slides were incubated overnight at 4 ꢀC with the primary antibody
Flk-1 (1:80) or anti-CD31 Class II (1:50) for the determination of
VEGF receptor type-2 or microvessel density, respectively. After
conjugation with streptavidinebiotineperoxidase complex,
coloring was performed with 3,30-diaminobenzidine substrate-
chromogen and Mayer’s hematoxylin was used for counter staining.
triplicate wells prepared for each individual dose, monolayer cells
incubated with the compounds for 48 h at 37 ꢀC and in atmosphere
of 5% CO2. After 48 h, cell were fixed, washed and stained with
Sulfo-Rhodamine-B stain, excess stain was washed with acetic acid
and attached stain was recovered with TriseEDTA buffer, color
intensity was measured in ELISA reader.
The relation between surviving fraction and complexes
concentration were plotted to get the survival curve for breast
tumor cell line after the specified time. The molar concentration
required for 50% inhibition of cell viability (IC50) was calculated and
compared to the reference drug Cisplatin (CAS, 15663-27-1). The
surviving fractions were expressed as means ꢂ standard error.
2.7.7. Tumor mass
2.7. In vivo anti-tumor activity
All tumors, 2-discs/animal, were punched out, weighed imme-
diately, and the average weight was calculated. All tumors were then
kept in 10% neutral buffered formalin for histological evaluation.
2.7.1. Experimental animals
All animal procedures and experimental protocols were
approved by the research ethics committee of Suez CanalUniversity
and were carried out in accordance with the guide for the care and
use of laboratory animals. Swiss albino mice weighing 20e25 g
were obtained from the Egyptian organization for biological
products and vaccines (Vacsera, Egypt). They were housed under
controlled conditioning (25 ꢂ 1 ꢀC constant temperature, 55%
relative humidity, and 12 h dark/light cycles), food and water
were allowed ad libitum throughout the study period.
2.8. Quantification of immunohistochemistry
For quantitative analysis, the intensities of immunoreactive
parts were measured. Measurements were done using an image
analyzer (ImageJ programme). For each slide from all experimental
groups, 7 fields were randomly selected; integrated densities
(IntDen) of nine random parts within each field were analyzed and
the mean was expressed as field IntDen.
2.7.2. Induction of solid tumors
2.9. Data analysis
EAC cells were suspended in normal saline so that each 100 ml
contains 5 ꢃ 106 cells. Each mouse was inoculated intradermally
Statistics were calculated with SPSS for windows version 14.0,
the mean values obtained in the different groups were compared by
one way ANOVA followed by Duncan’s multiple range test. All
results were expressed as mean values ꢂ SE and significance was
defined as p ꢄ 0.05.
(i.d.) at 2 sites bilaterally on the lower ventral side with 100 ml EAC
suspension (2.5 ꢃ 106 cells) on each site [41].
2.7.3. Induction of ascites
In a parallel experiment, mice were injected intraperitoneally
(i.p.) with 100
m
l EAC suspension (5 ꢃ 106 cells)/mouse to induce
2.10. Test of anti-bacterial activity
ascites. These mice received the same pharmacological treatment
as explained below [42e45]. At the end of the experiment, the
ascites fluids were collected and divided into two portions, the 1st
portion was immediately snap frozen in liquid nitrogen for the DNA
extraction and the other portion was used for the determination of
EAC cell counts.
In vitro anti-bacterial activity of 2-thioacetic acid benzothiazole
and its europium (III)- and terbium (III)-2-thioacetate benzothia-
zole complexes were studied by using the minimum inhibitory
concentration (MIC) method [47e49] against two pathogenic
strains; Escherichia coli and Salmonella spp.
All the bacteria were incubated and activated at 37 ꢀC by inoc-
ulation into Mueller-Hinton Broth for 24 h. The compounds were
dissolved in DMSO and then diluted using Mueller-Hinton Broth.
Different serial concentrations of the compounds were used to
determine the minimum inhibitory concentration (MIC) ranging
2.7.4. Pharmacological treatment
The mice were randomly assigned to 3 main groups, 8 mice
each. The first group received vehicle injection (DMSO in PBS) and
served as control. Second and third groups were injected daily with
4 mg/kg (i.p.) of europium (III)-2- benzothiazole or terbium (III)-2-
benzothiazole complex, respectively. All treatments started 6 days
after tumor cells inoculation [42e45]. At the end of the experiment,
mice were sacrificed by cervical dislocation then tumors were
excised and fixed in 10% neutral buffered formalin.
from 2500
The lowest concentrations of antimicrobial agents that resulted in
mM to 1 m
M. Test cultures were incubated at 37 ꢀC (24 h).
complete inhibition of growth were represented as MIC (
mM).
3. Results and discussion
2.7.5. DNA extraction
3.1. Chemistry of the complexes
DNA was purified from EAC cells using the GF-1 DNA extraction
kit (Vivantis). DNA fragments were analyzed in the automatic multi-
capillary electrophoresis QIAxcel system. Isolated DNA was placed
Analytical data for the synthesized Ln(III)-2-thioacetate benzo-
thiazole complexes are presented in Table 1. The elemental
analytical data show that the formulas of the complexes could be
[Ln(TAB)2Cl.3H2O].5H2O as displayed in Scheme 1. The complexes
in the instrument sample tray, 10
ml of the DNA samples were
automatically injected into the capillary channel and subjected to

102
B.H.M. Hussein et al. / European Journal of Medicinal Chemistry 51 (2012) 99e109
Table 1
Elemental analytical data for the ligand and Ln(III)-complexes.
Complex
C (%) found (calc.)
H (%) found (calc.)
N (%) found (calc.)
S (%) found (calc.)
Ln (%) found (calc.)
2-Thioacetic acid benzothiazole
[Tb (TAB)2Cl.3H2O].5H2O
[Eu(TAB)2Cl.3H2O].5H2O
49.0 (47.98)
26.9 (27.47)
28.3 (27.71)
3.40 (3.13)
3.99 (3.85)
3.29 (3.88)
6.23 (6.22)
4.03 (3.56)
4.09 (3.59)
28.1 (28.4)
8.0 (8.15)
7.61 (8.22)
e
19.8 (20.19)
19.2 (19.48)
are yellowish brown colored and stable. They are soluble in DMF
and DMSO and insoluble in water, ethanol, benzene, diethyl ether
and tetrahydrofuran. Because of the insolubility of the complex in
suitable solvents we were unsuccessful in growing crystals for
single crystal X-ray structural studies.
The luminescence properties of Eu (III) and Tb (III)-complexes
were measured at room temperature as shown in Figs. 1 and 2.
Owing to the solubility of the complexes, the luminescence spectra
of the complexes have been investigated in DMSO. Luminescence
spectrum of Eu(III)-2-thioacetate benzothiazole complex shows
the characteristic emission bands for Eu (III) ions. The emission
band centered at 615 nm (5D0/7F2) is obviously higher than
the emission band 590 nm (5D0/7F1). The luminescence spectrum
of Tb(III)-(TAB)2 shows the emission bands at 490, 545, 582,
and 618 nm corresponding to the 5D4/7F6, 5D4/7F5, 5D4/7F4,
5D4/7F3 transition respectively, and the strongest among them is
5D4/7F5.
The FTIR spectrum of the ligand shows a characteristic stretching
absorption bands at 3435,1710,1573,1027 and 694 cmꢁ1 assigned to
the stretching of hydroxyl, carbonyl, C]N of the thiazole ring, CeO
of the hydroxyl in the carboxylate group and the stretching of CeS
group [39], respectively. On complexation these bands were shif-
ted to a lower or higher frequency region. This shift is probably due
to the complexation of the lanthanide (III) ions to the ligand through
oxygen of the carbonyl group. Stretching of Ln(III)-oxygen bands of
the complexes appeared in low frequency region (515e530 cmꢁ1).
In the mass spectra of the complexes, the molecular ion peaks
(Mþ.) are observed at 780.1 and 787 m/z which are ascribed to [Eu
(TAB)2Cl.3H2O].5H2O and [Tb(TAB)2Cl.3H2O].5H2O, respectively.
The fragment ion peaks observed at 600 and 607 m/z are due to Eu
(TAB)2 and Tb (TAB)2 respectively.
3.2. DNA binding activity
Numerous biological experiments have demonstrated that DNA
is the primary intracellular target of anticancer drugs due to the
interaction between small molecules and DNA, which cause DNA
140
70
0
140
70
0
400
500
400
500
Wavelength(nm)
Wavelength(nm)
5
40
30
20
10
D
80
40
0
4
5
D
5
D
0
4
7
F
7
6
F
5
D
5
D
7
5
D
5
F
2
0
0
0
7
7
7
F
F
1
F
3
4
5
5
D
D
4
4
7
7
F
F
4
3
480
540
600
560
630
700
Wavelength(nm)
Wavelength(nm)
Fig. 1. Fluorescence emission spectra of Tb(III)-complex. Conditions: in DMSO, 25 ꢀC,
1 ꢃ 10ꢁ3 M, lex ¼ 342 nm, emission and excitation slit width were 5 nm.
Fig. 2. Fluorescence emission spectra of Eu(III)-complex. Conditions: in DMSO, 25 ꢀC,
1 ꢃ 10ꢁ3 M, lex ¼ 340 nm, emission and excitation slit width were 5 nm.

B.H.M. Hussein et al. / European Journal of Medicinal Chemistry 51 (2012) 99e109
103
damage in cancer cells, blocking the division of cancer cells and
resulting in cell death [50,51]. In this work the interactions of Eu(III)
or Tb(III)-2-thioacetate benzothiazole complexes with DNA were
investigated by fluorescent and UV spectroscopy.
The binding constants (K) of the complexes with DNA can be
calculated from the absorption titration experiments according to
following equation [35]
1
1
1
¼
þ
(1)
D
εa
D
ε
D
εK½DNAꢅ
3.2.1. Electronic absorption spectroscopy
Electronic absorption spectroscopy is employed to identify the
binding mode of DNA with the metal complex. Three fundamentally
different modes of DNA binding by metal complex can be identified:
non-specific external association, groove binding and intercalative
binding that small molecules intercalate into the base pairs of
nucleic acids [52,53].
The electronic absorption spectra of the Eu (III) and Tb(III)-
complexes with or without ct-DNA are shown in Fig. 3. The elec-
tronic absorption spectra of complexes consist of three resolved
bands at about 281, 290 and 300 nm. The high energy absorption
band appeared in the spectra of respective complexes are assigned
where
D
εa ¼ (εa ꢁεf) and
D
ε ¼ (εb ꢁεf). εa, εf and εb are the apparent
extinction coefficients of complex, the extinction coefficient for the
free complex, and the extinction coefficient for the complex in the
bound form, respectively. The double reciprocal plots of 1/Dεa
versus 1/[DNA] were linear (Fig. 4) and the binding constants were
calculated from the ratio of the slope to the y-intercept. The binding
constants obtained for Eu(III) and Tb(III)-complexes to DNA are
2.55 ꢃ 105 and 9.72 ꢃ 104 Mꢁ1, respectively. These binding constant
values compare well with that obtained for the intercalation of
complex into the DNA molecules [54e57].
to
p-p* transitions. Upon the addition of DNA, the above bands
3.2.2. Luminescence titration with ct-DNA
corresponding to complexes showed significant hypochromism
accompanied with slight red shift from 381 to 383 nm. These
results are similar to those reported earlier for various intercala-
tions [52], suggesting that the Ln(III)-complexes showed strong
binding to DNA via an intercalative mode.
The interaction of Eu(III)- and Tb(III)-2-thioacetate benzothiazole
with DNA in TriseHCl buffer at pH 7.4 were investigated by fluo-
rescence measurement as shown in Figs. 5 and 6. The effect of DNA
concentrations on the fluorescence intensity of Ln (III) exhibited
a pronounced change in emission intensity. The fluorescence
A
A
a
f
0.5
0.0
250
275
300
325
Wavelength (nm)
a
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
-0.1
B
B
h
260
280
300
320
340
Wavelength (nm)
Fig. 3. Electronic absorption spectra of Eu(III)-complex (A) and Tb(III)-complex (B) in
the absence and the presence of increasing amounts of ct-DNA. Arrows show the
absorbance changes upon increasing ct-DNA concentration.
Fig. 4. Double reciprocal plots for the effect of ct-DNA concentration on the absorption
spectra of Eu(III)-complex (A) and Tb(III)-complex (B) at room temperature.

104
B.H.M. Hussein et al. / European Journal of Medicinal Chemistry 51 (2012) 99e109
7.5
a
f
f
50
a
7.0
6.5
0
400
450
500
540
570
600
630
Wavelength (nm)
Wavelength (nm)
Fig. 5. Fluorescence emission spectra of Eu(III)-complex in the absence and in the presence of DNA. Conditions: in TriseHCl buffer pH 7.4, 25 ꢀC, Eu(III)-complex at 2 ꢃ 10ꢁ5 M,
lex ¼ 340 nm, emission and excitation slit width were 5 nm.
intensity of Ln(III) in the complexes enhanced with increasing DNA
concentration, while the fluorescence intensity at 415 in the region
of ligand decreases with increasing of DNA concentration due to the
interaction of DNA with Ln(III)-complexes.
In order to compare quantitatively the binding strength of the
complexes, the binding constants of them with ct-DNA were
obtained by monitoring the changes in the fluorescence intensities
of Eu(III)- and Tb(III)-complexes at 614 and 545 nm, respectively,
with increasing concentration of DNA using the following equation
[58,59]:
signs for free energy DG (30.58 and 28.16 kcal/mol for Eu(III) and
Tb(III)-complexes, respectively) mean that the interaction process
of Ln(III)-complex and DNA is spontaneous. The binding constants
of the formed complexes (K) between the Ln(III)-complex and DNA
are essentially of the same order of magnitude as that of the well-
known intercalators [53e57].The values of binding constant
revealed that the Eu(III)-complex is strongly bound with ct-DNA
than Tb(III)-complex.
3.3. In vivo anti-tumor activity
ꢀ
ꢁ
F ꢁ F0
log
¼ logK þ n log½DNAꢅ
(2)
3.3.1. Anti-angiogenic activity
F
To investigate the effect of our compounds on the tumor
angiogenesis, we evaluated the expression of CD31 and VEGF
receptor type-2 (Flk-1). CD31 is commonly used as an endothelial
cell marker to highlight tumor blood vessels reflecting the degree of
angiogenesis [41,60e63]. EAC-control group showed the highest
number of microvessel density on the fourteenth day post-
inoculation. Groups treated with Eu(III)- or Tb(III)-2-thioacetate
benzothiazole showed a significant reduction (P ꢄ 0.05) in micro-
vessel density on the fourteenth day compared to the control group
(Fig. 8). These results indicate that Eu(III)- and Tb(III)-2-thioacetic
acid benzothiazole have anti-angiogenic activity against EACs
(Fig. 9).
Where K and n are the binding constant and the molar ratio,
respectively. [DNA] is the free concentration of DNA. The Plot of log
[(FꢁF0)/F] versus log [DNA] at room temperature gave a straight
line (Fig. 7). The slope of such curve is equal to n while the intercept
is equal to log K. The values of n approximately equal to 1, indicating
that there is one binding site in Eu(III) and Tb(III)-complexes for
DNA. Where the magnitudes of binding constants (K) were calcu-
lated to be 2.296 ꢃ 105 Mꢁ1 and 8.616 ꢃ 104 Mꢁ1 corresponding to
Eu(III) and Tb(III)-complexes, respectively. These values are very
comparable to that calculated from the absorption titration with
correlation coefficient of 0.99. It was observed that the negative
a
f
10
f
a
80
8
40
520
560
600
640
400
450
500
Wavelength (nm)
Wavelength (nm)
Fig. 6. Fluorescence emission spectra of Tb(III)-complex in the absence and in the presence of ct-DNA. Conditions: in TriseHCl buffer pH 7.4, 25 ꢀC, Tb(III)- complex at 2 ꢃ 10ꢁ5 M,
lex ¼ 342 nm, emission and excitation slit width were 5 nm.

B.H.M. Hussein et al. / European Journal of Medicinal Chemistry 51 (2012) 99e109
105
120
100
80
A
-1.1
-1.2
-1.3
-1.4
-1.5
-1.6
60
*
*
40
20
0
Control
Eu-complex
Tb-complex
Fig. 9. Statistical analysis of the Eu(III)- and Tb(III)-2-thioacetate benzothiazole
complexes treatment on CD31 assessed immunohistochemically on day 14 post-
implantation of EAC cells in Swiss albino mice.
-5.9
-5.8
-5.7
-5.6
-5.5
-5.4
-5.3
-5.2
expression along with high VEGF receptor expression is associated
with increased microvessel density, poor clinical prognosis, and
increased metastasis in many cancers [41,67,68]. The loss of VEGF
expression in a tumor causes a dramatic decrease in vascular density
and vascular permeability and increases tumor cell apoptosis [69].
Strong intra-tumoral expression of Flk-1 was observed in all
EACs in the control group (Fig. 10). Flk-1-rich tumors were detected
in untreated EACs while the groups treated with Eu(III)- or Tb(III)-
2-thioacetate benzothiazole complex showed poor expression of
the receptor with significant reduction (P ꢄ 0.05) in the integrated
density as compared to the untreated control (Fig. 11).
log [DNA]
B
-0.7
-0.8
-0.9
-1.0
-1.1
-1.2
-1.3
3.3.2. Tumor mass and number of EAC cells
To determine the in vivo effect on tumor cell growth, mouse
mammary carcinoma cell line EAC was treated with complexes
europium(III)- and terbium(III)-2-thioacetate benzothiazole
(4 mg/kg body weight/ip), daily for total of seven doses during its
growth period, starting at the day 6 after inoculation of tumor
cells. Solid tumor mass and number of EAC cells were evaluated at
the end of treatment. In control EAC-bearing mice, there was an
increase in solid tumor mass and number of EAC cells. A rise in
solid tumor weight of about 165 mg over a period of 14 days of
tumor growth was observed. However, when treated with the
[Europium(III)-2-thioacetate benzothiazole] and [Terbium(III)-2-
thioacetate benzothiazole] complexes, about 39% and 52%
decrease in the solid tumor mass was observed, respectively,
indicating the anti-tumor effect of [Europium(III)-2-thioacetate
benzothiazole] and [Terbium(III)-2-thioacetate benzothiazole]
complexes as shown in Fig. 12.
-5.9
-5.8
-5.7
-5.6
-5.5
-5.4
-5.3
-5.2
log [DNA]
Fig. 7. Double logarithm plots for the effect of ct-DNA concentration on the fluores-
cence intensity of Eu(III)-complex (A) and Tb(III)-complex (B) at room temperature.
VEGF and its receptors have been implicated as key
components in the vascularization of a tumor [41,64]. But, most of
the VEGF angiogenic properties are mediated by interaction with
VEGF receptor type-2 (Flk-1) [65,66]. Studies show that high VEGF
Similarly, single treatment with Eu(III)- or Tb(III)-2-thioacetate
benzothiazole complexes produced
a
significant reduction
Fig. 8. Effect of treatment of Eu(III)- and Tb(III)-2-thioacetate benzothiazole complexes on CD31 assessed immunohistochemically on day 14 post-inoculation of EAC cells in Swiss
albino mice (A) control; (B) Eu(III)-2- thioacetate benzothiazole treatment; (C) Tb(III)-2- thioacetate benzothiazole treatment.

106
B.H.M. Hussein et al. / European Journal of Medicinal Chemistry 51 (2012) 99e109
Fig. 10. Effect of treatment of Eu(III)- and Tb(III)-2-thioacetate benzothiazole complexes on VEGF assessed immunohistochemically on day 14 post-inoculation of EAC cells in Swiss
albino mice (A) control; (B) Eu(III)-2-thioacetate benzothiazole treatment; (C) Tb(III)-2-thioacetate benzothiazole.
120
100
80
60
40
20
0
Control
Eu-complex
Tb-complex
Fig. 11. Statistical analysis of Eu(III)- and Tb(III)-2-thioacetate benzothiazole
complexes treatment on VEGF assessed immunohistochemically on day 14
post-inoculation of EAC cells in Swiss albino mice.
Fig. 13. Statistical analysis of the Eu(III)- and Tb(III)-2-thioacetate benzothiazole
complexes treatment on number of Ehrlich ascites carcinoma cells (or EAC).
(P ꢄ 0.05) in number of EAC cells in ascetic fluid on day 14 post-
inoculation as compared to the control animals (Fig. 13).
cleavage of the cell’s DNA at intervals of 160e240 base pairs (bp)
which are the size of oligonucleosomes [70e72].
3.3.3. DNA fragmentation assay
An attempt was made to find the mechanism of inhibition of
proliferation of EAC cells by Eu(III)- and Tb(III)-2-thioacetate ben-
zothiazole complexes. As expected, DNA fragments were observed
in EAC cells after treatment with Eu(III)- and Tb(III)-2-thioacetic
acid benzothiazole complexes (Fig. 14. lane B & C) and no frag-
mentation was observed in untreated EAC cells (Fig.14. lane A). This
indicates that there is apoptotic role of Eu(III) and Tb(III)-2-
thioacetate benzothiazole complexes in EAC cells.
The oligonucleosomes fragments can be observed as a DNA
ladder on agarose gel electrophoresis. Limited information about
DNA fragmentation has been obtained by traditional agarose gel
electrophoresis. Therefore, the capillary electrophoresis has been
conducted to analysis the DNA fragments. Capillary electrophoresis
(CE) has been widely applied to DNA separation and characteriza-
tion of cell apoptosis based on the determination of very small DNA
fragmentation from 15 bp with a size difference of 5 bp [73,74].
Figs. 15 and 16 show the data of DNA fragments extracted from
EAC cells treated by Eu(III)- and Tb(III)-2-thioacetic acid benzo-
thiazole complexes for 7 days. Many peaks appeared for DNA
treated with Eu(III)-complex as shown in Fig. 15. By comparing the
migration time of the peaks with those of the standard DNA ladder
marker 50e800 bp, peaks 1e5 have sizes of about 40, 69, 89, 108
and 121 bp which can be regarded as cleaved DNA fragments
of short length. Peaks 6e9 have approximately sizes of 151, 183, 203
and 219 bp, respectively, corresponding to one to four nucleosomes.
Fig.16 displays the data for DNA treated with Tb(III)-complex, by
comparing with the reference marker peaks 1e5 which have sizes
Activation of programmed cell death (apoptosis) in cancer cells
offers novel and potentially useful approaches to improve patient
responses to conventional chemotherapy. The most dramatic
biochemical feature of apoptosis is DNA fragmentation as a result of
200
180
160
140
120
100
80
60
40
20
0
Control
Eu-complex
Tb-complex
Fig. 12. Statistical analysis of the Eu(III)- and Tb(III)-2-thioacetate benzothiazole
complexes treatment on solid tumor mass.

B.H.M. Hussein et al. / European Journal of Medicinal Chemistry 51 (2012) 99e109
107
Mediterranean Region (EMR) especially in Egypt and breast cancer
which represents the most common cancer among women in Egypt,
representing 35.1% of total cancer cases [75].
Cisplatin, which is one of the most effective metallo-anticancer
agents, was used as the reference drug in this study. The relation-
ship between surviving fraction and drug concentration was plotted
to obtain the survival curve of HepG2 and MCF7 cell lines. The
responseparametercalculatedwastheIC50 value, whichcorresponds
to the concentration required for 50% inhibition of cell viability.
As shown in Fig. 17, [Eu(III)-2-thioacetate benzothiazole]
and [Tb(III)-2-thioacetate benzothiazole] complexes inhibited
the growth MCF7 cell line with IC50 values of 5.1 ꢂ 0.2 and
4.5 ꢂ 0.15
which is about 2 times the strength of cisplatin (IC50 values of
M).
mM, respectively. Complexesed show better cytotoxicity
9.0 ꢂ 0.28
m
Fig.18 displays the effect of [Eu(III)-2-thioacetate benzothiazole]
and [Tb(III)-2- thioacetate benzothiazole] complexes on HepG2
cell growth. The antiproliferation effect of the [Eu(III)- and
Tb(III)-2-thioacetate benzothiazole] complexes revealed that
[Eu(III)-complex] IC50 ¼ 25 ꢂ 0.17
mM and [Tb(III)-complex]
IC50 ¼ 35 ꢂ 0.2
mM.
Table 2 shows the in vitro cytotoxic activity of the synthesized
compounds. It can be seen that Tb(III)-complex was the most potent
compound in this screening, and exhibited a higher cytotoxic
Fig. 14. Effect of Eu(III)- and Tb(III)-2-thioacetate benzothiazole complexes on
genomic DNA of EAC cells: EAC cells treated with or without Eu(III), Tb(III)-2-
thioacetate benzothiazole complexes in vivo were harvested and DNA was isolated;
Lane 1: DNA of EAC untreated, Lane 2: DNA of EAC treated with Eu(III)-2-thioacetate
benzothiazole complexes Lane 3: DNA of EAC treated with Tb(III)-2-thioacetate ben-
zothiazole complexes.
activity (IC50 ¼ 4.5
reference drug cisplatin (IC50 ¼ 9
with IC50 ¼ 5.1 M. On the otherhand, Eu(III)- and Tb(III)-complexes
showed IC50 values of 25 M and 35 M, respectively, which are
m
M) against MCF7 when compared with the
mM), followed by Eu(III)-complex
m
m
m
lower than that of cisplatin (IC50 ¼ 12 ꢂ 0.88
mM), indicating lower
activity against HepG2 cell line.
of about 47, 72, 91, 112 and 125 bp which can be regarded as cleaved
DNA fragments of short length. Peaks 6e11 have approximately
sizes of 152, 170, 192, 210, 231 and 317 bp, respectively, corre-
sponding to one to eight nucleosomes.
The biochemical character of apoptosis is the cleavage of
genomic DNA into 160e240 bp fragments [70,71]. Therefore peaks
6e9 for DNA fragments treated with Eu(III)-complex and peaks
6e11 for DNA fragments treated with Tb(III)-complex can be
regarded as typical apoptotic DNA fragments, suggesting that the
cells undergo apoptosis after they have treated with Eu(III)- and
Tb(III)-2- thioacetate benzothiazole complexes for 7 days.
3.5. Antimicrobial activity
There is a need for new antimicrobial agents and one of the
attractive strategies in this field is to develop nucleic acid targeting
agents that inhibit the pathogenic gene to replicate and transcript.
The central dogma of all living organisms (prokaryotic and
Eukaryotic) is based on replication of DNA, transcription to produce
mRNA and translation to synthesis proteins. Inhibition of DNA
transcription and replication would restrict protein synthesis, or
replication, and could induce cell death. On the basis of a previously
observed correlation between the antimicrobial activity and
DNA binding strength of some molecules [76e81], the Eu(III)- and
Tb(III)-complexes were screened for anti-bacterial activity against
Escherichia coli and Salmonella by the minimum inhibitory
concentration (MIC) method [47e49].
3.4. In vitro anti-tumor activity
HepG2 and MCF7 cell lines were selected for preliminary studies
on the in vitro anti-tumor activity of the [Eu(III)-2-thioacetate ben-
zothiazole] and [Tb(III)-2-thioacetate benzothiazole] complexes.
Our choice was based on the fact that hepatocellular carcinoma
(HCC) being one of the most common and lethal types in Eastern
The results showed that the antimicrobial activity of the
complexes (Eu(III)- or Tb(III)-2-thioacetate benzothiazole) against
the Escherichia coli and Salmonella is much higher than
2-thioacetic acid benzothiazole. The results of our study showed
Fig. 15. Capillary electropherogram of DNA extracted from EAC cells after treatment with Eu(III)-2-thioacetate benzothiazole complex in vivo.

108
B.H.M. Hussein et al. / European Journal of Medicinal Chemistry 51 (2012) 99e109
Fig. 16. Capillary electropherogram of DNA extracted from EAC cells after treatment with Tb(III)-2-thioacetate benzothiazole complex in vivo.
mechanism was hypothesized that the benzothiazole complexes
has an effect on the two pathogenic bacteria cell proliferation.
10
9
8
7
6
5
4
3
2
1
0
9
4. Conclusion
5.1
4.5
Two new trivalent lanthanide complexes have been synthesized
and characterized using various spectroscopic methods which
revealed that the ligand forms mononuclear Eu(III)- and Tb(III)-
complexes with 1:2 stoichiometry between lanthanum ions and
ligand. The newly synthesized complexes were evaluated for DNA
binding, antimicrobial activity and cytotoxicity studies. We
conclude from the current study that there is a strong interaction
between Eu(III)- or Tb(III)-2-thioacetate benzothiazole complexes
and DNA by absorption and fluorescence spectral techniques which
is due to the intercalation into the hydrophobic state of the ds-DNA
Cisplatin
Eu-complex
Tb-complex
M). IC50 values
Fig. 17. IC50 values for Eu(III)- and Tb(III)-complexes against HepG2 (
are presented as the mean ꢂ SE obtained from at least three independent experiments.
m
with binding constants ranging from 103-105 Mꢁ1
.
Furthermore thepositive results presented in this work regarding
the anti-bacterial and anti-tumor activity of Eu(III) and Tb(III)-2-
thioacetate benzothiazole complexes confirm our preliminary
hypothesis of strong interaction proposed early in this paper.
In addition, our novel compounds proved to exert an anti-
angiogenic effect as it was evident by the reduction of micro-
vessel density through, at least in part, the inhibition of VEGFR type
2. The apoptotic study indicates that the two complexes can
effectively induce the apoptosis of EAC cells by formation inter-
nucleosomal DNA ladder. Moreover Eu(III)- and Tb(III)-2-thioacetic
benzothiazole acid complexes exhibited significant anticancer
activity, when compared to cisplatin as a reference drug. The ach-
ieved results suggest that these synthetic compounds have a strong
therapeutic potential as anti-tumor and anti-bacterial agents.
40
35
35
30
24
25
20
15
10
5
12
0
Cisplatin
Eu-complex
Tb-complex
M). IC50 values
Fig. 18. IC50 values for Eu(III)- and Tb(III)-complexes against MCF7 (
m
are presented as the mean ꢂ SE obtained from at least three independent experiments.
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